Lightcycler relative quantification software

To measure TMEM240 mRNA expression, real-time reverse transcription PCR (RT–PCR) was performed in a LightCycler 96 (Roche Applied Science, Penzberg, Germany). Real-time PCR was performed using the SensiFAST™ Probe No-ROX Kit (Bioline, London, UK, Cat. No. BIO-86020) with specific primers and the corresponding Universal Probe Library probe (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instructions. The glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) was used as a reference gene. The PCR conditions were as follows: preincubation at 95 °C for 10 min followed by 40 cycles of amplification at 95 °C for 10 s and 60 °C for 10 s. The normalized gene expression values obtained using LightCycler Relative Quantification software (Version 1.5, Roche Applied Science) were compared with those of the control group. TMEM240 mRNA expression was considered low if the mRNA expression level of TMEM240 relative to GAPDH was 0.5-fold lower in the breast tumor tissue than in the paired normal breast tissue. The primers and probes used in RT–PCR are listed in Additional file 1: Table S1.

The Ambion Cells-to-cDNA™ II Kit (AM 1723, Life technologies, UK) was used for the isolation of total RNA and cDNA synthesis from cells of each zone according to the manufacturer's instructions. Quantitative RT-PCR is an accurate way of both detecting and quantifying a given DNA sequence and represents the most sensitive way of detecting differences in mRNA expression irrespective of exact cell number. The standard reference gene used for the studies below was GAPDH. The primers for qRT-PCR are listed in Table 1. Quantitative RT-PCR was performed using the LightCycler™ quantitative PCR machine (Roche, Switzerland). Each reaction was composed with 5 μl master mix, 3.7 μl RNase-free water, 0.4 μl forward and reverse primer mix, 0.8 μl template cDNA and 0.1 μl COX (all reagents from Promega, UK). Quantitative RT-PCR was performed using the LightCycler at 95 °C for 5 min, followed by 40 cycles of 94 °C for 15 s, primer specific annealing temperature for 30 s, and 72 °C for 20s, with a single data acquisition step. The crossing point for each transcript was determined using the LightCycler software (Roche, Switzerland). The LightCycler Relative Quantification software (Roche, Switzerland) was used to analyse the data. Each cDNA sample was measured in triplicate with GAPDH as the reference gene and using RNAse-free water as the negative control.

Visceral adipose tissue was isolated, immediately frozen in liquid nitrogen and then grinded. Adipocytes and total bone marrow isolates were immediately lysed using the TriPure^® Isolation Reagent (Roche). Samples were stored at -80°C until RNA preparation. Total RNA was extracted according to the manufacturer’s protocol. Following DNase treatment, cDNA was synthesized using the QuantiTect^® Reverse Transcription Kit (Quiagen) according to the manufacturer’s instructions. Relative expression levels of per2, reverbα, cxcl12, tnfα, il1β, il18, nlrp3, leptin and adipsin were measured in a Light Cycler^® 480 system (Roche) using TaqMan hydrolysis probes (see Supplementary Table 1). Rplp0 was used as a reference. The second derivate maximum method was applied for data analysis using LightCycler^® Relative Quantification Software (version 1.5.0.39, Roche).

The DNA methylation level of SMAD3 was gauged using TaqMan QMSP with light cycler 480 (Roche Applied Science) after the bisulfite conversion with the EpiTect Fast DNA Bisulfite Kit (Qiagen, Bonn, Germany, cat. no. 59826), which was suggested by the manufacturer. To perform QMSP, a SensiFAST™ Probe No-ROX Kit (Bioline, London, UK; Cat. no. BIO-86020) with specific primers and probe was used for SMAD3. Normalized DNA methylation values from LightCycler Relative Quantification software (version 1.5, Roche Applied Science) were compared with the control group. The relative SMAD3 DNA methylation level was normalized to Beta-actin (ACTB). ACTB can work as total genomic DNA or ccfDNA content control. SMAD3 was considered hypomethylated when the methylation level of SMAD3 relative to that of the ACTB gene was half that in CRC tissue compared with the paired noncancerous colorectal tissue sample. In circulating methylation, the average value of SMAD3 relative to that of the ACTB gene in healthy tissue was 25.37. In CRC, a value of less than 0.5 (50-fold lower in CRC than healthy tissue) was regarded as hypomethylation. Table 3 presents the primers. The specificity of SMAD3 methylation end products was confirmed by bisulfite sequencing (Figure S3).

Total cellular RNA was extracted using Trizol reagent and followed by complementary DNA (cDNA) synthesis. The expression of each sample was normalized for RNA preparation and RT reaction on the basis of its glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA content [26] (link). For detection of the amplification products in GAPDH and gelsolin RT-PCR, we used a LightCycler-FastStart DNA Master SYBR Green I Kit (Roche Molecular Biochemicals, Penzberg, Germany). Thermal cycling started with 10 min at 95°C, followed by 30 cycles of 95°C for 15 s, 68°C for 10 s and 72°C for 15 s (amplification product data acquisition at 86°C). For amplification and detection, we used LightCycler Relative Quantification Software (Roche Molecular Biochemicals). To determine the RT-PCR efficiency (E), we analyzed a serial dilution of a GAPDH and gelsolin cDNA over the range in which we measured cDNA. In this range, the PCR efficiency (E) for both genes was 1.88. The relative gelsolin to GAPDH expression was calculated using the Delta-Cp approach based on the expression 1.88 – Delta- Cp. The following sequence-specific primers (MWG Biotech, Ebersberg, Germany) were used:
GAPDH forward, 5′-AGATTGTCAGCAATGCATCCTGC-3′;
GAPDH reverse, 5′-CCTTCTTGATGTCATCATACTTGG-3′;
Gelsolin forward, 5′- CAGCCTCTGACTTCATCTCCAAG-3′;
Gelsolin reverse, 5′-CACGTTGGCAATGTGGCTGGAG-3′.